Metal ions play a crucial role in RNA function. Stabilization of the proper RNA tertiary structure critically depends on specific and nonspecific interactions with metal ions. The HDV ribozyme from the pathogenic hepatitis delta virus (HDV) is the only one of the four naturally occurring so-called small ribozymes that has relevance for human disease. Its metal ion specificity is low as self-cleavage of the HDV ribozyme is efficient in the presence of a variety of divalent (and monovalent) metal ions. To further examine the roles of divalent metal ions in folding and catalysis of the HDV ribozyme, I will conduct a set of new experiments that answer three fundamental questions: First, how do metal ions contribute to the folding of RNA into a three-dimensional structure? Second, how does this structure result in the acquisition of catalytic activity? Third, what role do metal ions play in conformational dynamics? The HDV ribozyme is well suited for such experiments, since it is small in size yet complex enough so that significant conformational changes occur during folding and catalysis. Specific Aims for this proposal are: (1) Correlate divalent metal ion interactions with local conformational changes in the trans-acting HDV ribozyme, and (2) investigate global folding transitions and conformational dynamics of the cis-acting HDV ribozyme, up to the single-molecule level. The importance of this work includes separating the roles of metal ions in HDV ribozyme function and improving our understanding of the contribution of metal ions to the dynamics and resulting catalytic activity of RNA enzymes in general. [unreadable] [unreadable]